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Analysis of in vitro micronucleus assays using the IN Cell Analyzer 3000 Micronuclei Formation Analysis Module

Key words: Micronucleus assays • toxicology • image analysis • IN Cell Analyzer


Micronucleus induction is a key characteristic of genotoxic compounds and analysis of micronuclei formation resulting from DNA strand breakage (clastogens) or interference with chromosome segregation (aneugens) is an important component of toxicology screening of new drug candidates. Manual scoring of micronucleus assays is time consuming and subject to operator variance, bias, and error while automated analysis of micronucleus assays is significantly faster and enables consistently objective scoring.

This application note describes the use of the IN Cell Analyzer 3000 Micronuclei Formation Analysis Module for automated scoring of micronucleus assays.


Materials
Products used

IN Cell Analyzer 3000 25-8010-11

IN Cell Analyzer 3000 Seat License* 63-0055-97

IN Cell Analyzer 1000 25-8010-26

IN Cell Analyzer 1000 Seat License* 25-8098-22

Micronuclei Formation Analysis Module28-4047-13

IN Cell Translator Image Converter 28-4047-40


Other materials required
CHO-KI cells (ATCC or ECACC)

Tissue culture reagents (Invitrogen)

Imaging grade 96-well plates (Greiner Bio-One or Perkin Elmer)

Mitomycin C (Sigma, M4287)

Cytochalasin B§ (Sigma, C6762)

Hoechst 33342 (Molecular Probes)

FITC (Molecular Probes, F1906)

* A seat license is a cost-effective single-user or server license that gives access to all ready to use Image Analysis Modules provided for your IN Cell Analyzer instrument. License holders have access to all appropriate analysis software and more licenses can be purchased as the number of users grows.

† Available to seat license holders only.

‡ Mitomycin C is classified as toxic; handle in accordance with product safety data sheets and local laboratory safety guidelines.

§ Cytochalasin B is classified as very toxic; handle in accordance with product safety data sheets and local laboratory safety guidelines.


Methods
Assay protocol
1. Seed CHO-K1 cells in imaging grade 96-well plates at 5000 cells/100 μl/well and incubate under standard tissue culture conditions for 24 h.

2. Prepare dilutions of test compounds in solvent. Prepare a 1-mg/ml stock solution of mitomycin C in PBS and sterile filter for use as a positive control. Add 100 μl of test compounds, solvent, and mitomycin C (use mitomycin C at 100-ng/ml final concentration) and incubate cells for 24 h.

3. Prepare a 3-mg/ml stock solution of cytochalasin B in DMSO. Dilute 1:100 in complete tissue culture medium and add 10-μl/well. Incubate cells for 24 h.

4. Remove media, wash cells once with PBS, and fix in ethanol for 30 min at room temperature.

5. Make 10-mg/ml stock solution of FITC in DMSO. Dilute 10 μl in 100-ml PBS and add 100-μl/well. Incubate for 30 min at room temperature.

6. Wash wells three times with PBS.

7. Stain cells with 5-μM Hoechst™ 33342 in PBS for 15 min at room temperature.

8. Image wells using excitation and emission settings for fluorescein and Hoechst 33342.


Analysis protocol
The IN Cell Analyzer 3000 Micronuclei Formation Analysis Module uses a series of operations based on user settings (Fig 2) to define nuclei, segregate mono-nucleate and binucleate cells, and define a search area for micronuclei (Fig 3). For full guidance on using the analysis module see the product manual.



1. Open run file in the IN Cell Analyzer 3000 analysis software.

2. Select a mitomycin C positive control well. Apply intensity threshold, erosion, and size filtration settings to identify nuclei in the blue (Hoechst) channel. Take care to exclude micronuclei from identified objects.

3. Adjust nuclear power (DNA content) and form factor (nuclear symmetry) settings to segregate mono-nucleate and bi-nucleate cells. Use delta threshold settings if required to assign ambiguous cells as unclassified.

4. Adjust threshold in the green (FITC) channel to identify cell boundaries.

5. Adjust search area settings to define regions to be analyzed for micronuclei.

6. Adjust intensity and size filtration settings to identify micronuclei.

7. Check analysis on second positive control well and solvent negative control wells. Adjust analysis parameters as necessary.

8. Run analysis.


Data analysis
1. Open the data output file and analyze micronuclei frequency (Fig 4) and proliferation index (Fig 5) data.

2. Discard data from wells where the cell number or the proliferation index indicate cytotoxic or cytostatic activity. Compounds showing potential false negative criteria should be re-tested at a lower concentration.

Data from typical assays carried out using the protocol described are shown in Figure 4. Exposure of cells to increasing concentrations of compounds of known genotoxicity results in an increase in the percentage of bi-nucleate cells with micronuclei. As cells are exposed to higher doses of compounds, cell cycle inhibition and cytotoxicity results in cell arrest prior to mitosis preventing micronuclei formation, with a resulting drop in micronuclei frequency at higher compound doses. This is evident from examination of proliferation index data (Fig 5), which reports the ratio of bi-nucleate to mono-nucleate cells.

Analysis of micronucleus assay data at compound concentrations below those which induce cell cycle arrest or cytotoxicity (Fig 6) allows determination of EC50 values for compounds in the in vitro micronucleus assay (Table 1).



Analysis of images acquired on IN Cell Analyzer 1000
Images acquired using IN Cell Analyzer 1000 may be analyzed using the IN Cell Analyzer 3000 Micronuclei Formation Analysis Module following conversion of images using IN Cell Translator Image Converter software (Fig 7). Converted images are analyzed using the analysis protocol described.


Conclusion
The IN Cell Analyzer 3000 Micronuclei Formation Analysis Module provides a rapid method (< 10 min for a 96-well plate) for analysis of in vitro micronucleus assays allowing automated screening of large numbers of compounds for genotoxicity. The software is compatible with standard assay protocols (1, 2) and may be used to analyze images acquired using the IN Cell Analyzer 1000 via image conversion to IN Cell Analyzer 3000 format.


References
1. Kirsch-Volders, M. et al. Report from the in vitro micronucleus assay working group. Mutat. Res. 540(2), 153–63 (2003).

2. Kirsch-Volders, M. et al. Report from the in vitro micronucleus assay working group. Environ. Mol. Mutagen. 35(3), 167–72 (2000).



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